Abstract
We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.
Highlights
The emergence and spread of new drug-resistant Staphylococcus aureus strains are a growing global concern, in hospital settings[1]
To retrieve information regarding the structural organization of LytU, we carried out an analysis of LytU primary structure and compared it to lysostaphin family members
We showed that the enzyme is bound to the plasma membrane and that the endopeptidase domain resides outwards the membrane
Summary
The emergence and spread of new drug-resistant Staphylococcus aureus strains are a growing global concern, in hospital settings[1]. The primary and key defence mechanism of the pathogen is to increase the number of peptidoglycan (PG) layers in its cell wall (CW)[2] Selective pressure driving this change results from the extensive use of the most prominent antistaphylococcal agents, beta-lactam antibiotics (e.g. methicillin) and glycopeptides (e.g. vancomycin) causing these antibiotics to become increasingly inefficient in combat against the new treatment-resistant strains[3]. The catalytic mechanism of the lysostaphin family enzymes has recently been investigated[13] It involves a zinc ion which polarizes the scissile peptide bond by coordinating the carbonyl oxygen, and two catalytic histidines which activate a water molecule or a hydroxide ion to act as a nucleophile to attack the carbonyl carbon. The in vitro active pH range for both LytM and lysostaphin is 5–914–16 and the optimum for lysostaphin is 7.516
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